Aberrant glycosylation patterns are a hallmark of the tumor phenotype. While highly variable in structure, many tumor-associated oligosaccharides share one important feature: they contain sialic acid residues. Indeed, the overexpression of sialic acid is highly correlated with the malignant phenotype in gastric, colon, pancreatic, liver, lung, prostate and breast cancers, as well as several types of leukemia. Consequently, new strategies for targeting cells on the basis of differential sialic acid expression levels may have widespread utility in the treatment and diagnosis of cancer. This proposal describes a chemicAL approach to the selective targeting of highly sialylated cells with therapeutic and diagnostic agents. The strategy is predicated on the remarkable tolerance of the sialic acid biosynthetic machinery for modified substrates. We have shown that a uniquely reactive functional group, the ketone, can be delivered to cell surface sialic acids by feeding the cells the unnatural metabolic precursor N-levulinoyl mannosamine (ManLev). The ketone provides the ideal mechanism for targeting cells in their native environment because it is chemically orthogonal to all other cell surface components, yet will react selectively with hydroxylamines and hydrazides under physiological conditions. Thus, in the context of the biological milieu, the ketone introduces a unique functional group which permits covalent targeting with molecules bearing complementary functionality. The objective of the proposed research is to explore the potential application of unnatural sialic acid biosynthesis to the selective delivery of therapeutic and diagnostic agents to human tumor cells. A positive correlation between sialic acid expression level and ManLev metabolism is critical for the proposed application, and will be established using tumor cell lines selected for defined sialic acid levels. Next, hydroxylamine-conjugated toxins, imaging reagents and small molecular antigens will be synthesized, and their selectivity for cells rich in sialic acid will be evaluated. As a prelude to future in vivo targeting studies, unnatural sialic acid biosynthesis in laboratory animals will be investigated. Finally, the biosynthetic pathway for cell surface fucosides will be explored as an alternative vehicle for the cell surface delivery of unique chemical targets. This project is the first phase of a long-term program focusing on applications of unnatural oligosaccharide biosynthesis.